Development and validation of a simple and …

1 Available online Journal of Chemical and Pharmaceutical Research, 2015, 7(8):774-782. ISSN : 0975-7384. Research Article CODEN(USA) : JCPRC5. Development and validation of a simple and sensitive stability indicating RP-HPLC assay method for determination of Nintedanib and stress degradation studies Dasari Purnachand1*, Arava Veerareddy1, Bhoomireddy Ramadevi2, Ch. V. S. L. Kameswarrao1, Gogireddy Surendra Reddy1 and Bethi Madhusudhanreddy1. 1. Research & Development Centre, Suven Life Sciences Limited, Hyderabad, India 2. Department of Chemistry, University, Hyderabad, India _____.

2 ABSTRACT. A Novel stability indicating Reverse Phase-Liquid Chromatography (RP-HPLC) method has been developed and validated for the determination of Assay of Nintedanib Drug Substance in the presence of degradation products generated from forced degradation studies. Chromatographic separation was achieved on YMC Pack ODS-AQ. (C18) column (Size: 250 x mm; 5 m particle size) at flow rate mL/min. with 210 nm detection. The mobile phase was Water: Acetonitrile (pH of water was adjusted to with Orthophosphoric acid) through gradient elution.

3 Product was subjected to stress conditions like acid, base, peroxide, thermal and photolytic degradation. No new impurities were observed during thermal and Photolytic degradation. Two new impurities were observed during acid degradation, one impurity was observed in base degradation and another impurity was observed in peroxide degradation. However, degradation products did not interfere with the detection of Nintedanib. Developed method was validated as per ICH guidelines using validation parameters like specificity, linearity, LOQ, accuracy, precision, robustness and ruggedness.

4 LOQ value was achieved at 2 g/mL concentration. Good linearity (r2 >. ) was obtained ranging from 25 g/mL to 150 g/mL concentrations. Recovery was verified by spiking 40. g/mL, 50 g/mL and 60 g/mL concentrated solutions to 50 g/mL concentrated solution. Hence newly developed RP-HPLC method is capable for estimating assay of Nintedanib Drug Substance and the present method is effectively separated the Nintedanib from its major degradation products. Keywords: Nintedanib, Development & validation , RP-HPLC, Assay, Degradation, LC-MS.

5 _____. INTRODUCTION. Nintedanib is being used to treat some types of non-small-cell lung cancer and for idiopathic pulmonary fibrosis (IPF). Nintedanib is a small molecule tyrosine-kinase inhibitor, targeting vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and platelet derived growth factor receptor (PDGFR). Nintedanib (formerly BIBF 1120) is originally developed by Boehringer Ingelheim and marketed under the brand names OFEV and VARGATEF [1-9]. Nintedanib (Mol. wt. ) is available as Esylate salt (Mol.)

6 Wt. ) in stable form. The Development and validation of the suitable analytical method for determination of Assay of Nintedanib Drug Substance (Fig. 1) was not reported in literature. It was felt essential to develop and validate a simple and sensitive stability indicating chromatographic method for determination of Assay of Nintedanib Drug Substance. Hence the objective of the current research was: (i) to study the degradation pattern of Nintedanib Drug Substance; (ii) to develop chromatographic stability indicating method for determination of Assay of Nintedanib Drug Substance; (iii).

7 774. Dasari Purnachand et al J. Chem. Pharm. Res., 2015, 7(8):774-782. _____. validation of the analytical method for quantitative determination of Nintedanib in accordance with the National and International guidelines [10-13]. Fig. 1 Chemical Structures of Nintedanib Esylate O. H. O N. O. NH. N. N O. N O S O. OH. Nintedanib Esylate C33H39N5O7S. Mol. Wt.: EXPERIMENTAL SECTION. Standards and Reagents: Nintedanib sample was obtained from In-house R&D laboratory of Suven Life Sciences. Nintedanib was well characterized by Infrared Spectroscopy (IR), Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR), Carbon Nuclear Magnetic Resonance Spectroscopy (13C-NMR), and Mass Spectroscopy.

8 Hydrochloric acid, Sodium Hydroxide, Hydrogen Peroxide, Ortho Phosphoric acid (OPA) of AR grade were obtained from RANKEM Chemicals. Acetonitrile of HPLC grade was obtained from RANKEM Chemicals. Water was used from Milli-Q water purification system (Make: MILLIPORE / Integral 5). Instrumentation and Software: The analytical method Development and validation is performed on High Performance Liquid Chromatograph (Make & Model: Shimadzu; Pump: LC-20AD XR, Auto Injector: SIL-20AC XR, PDA Detector: SPD-M20A) equipped with binary solvent delivery pump, degasser, auto-sampler and column thermostat using Empower Software (Version 3, Future release 1).

9 Chromatographic separation was achieved on YMC Pack C18 column (size: 250 x mm; 5 m particle size) using a gradient program at a flow rate of mL/min and an injection volume of 10 L with wavelength detection at 210. nm. Column oven temperature was set at 25 C. The mobile phase consists of water pH with orthophosphoric acid (MP-A) and Acetonitrile (MP-B) as per the gradient program given in Table 1. Table 1. HPLC Gradient Program Composition Time (min.) Mobile Phase-A Mobile Phase-B. (Water pH with OPA) (Acetonitrile). 00:00 50 50.

10 04:00 50 50. 05:00 0 100. 09:00 0 100. 09:10 50 50. 12:00 50 50. Preparation of Solutions Diluent / Blank Solution Acetonitrile:Water (90:10). 775. Dasari Purnachand et al J. Chem. Pharm. Res., 2015, 7(8):774-782. _____. Stock Solution An accurately weighed 25 mg of Nintedanib was transferred into 25 mL volumetric flask, sufficient amount of diluent was added to dissolve it and volume is made upto 25 mL to obtain 1000 g/mL concentrations. Standard Solution An aliquot of 5 mL from the Stock Solution was transferred into 50 mL volumetric flask, and volume is made upto 50 mL with diluent to obtain 100 g/mL concentrations.